Project Summary Autoreactive T cell responses target central nervous system-derived autoantigens and cause several demyelinating autoimmune diseases including multiple sclerosis, acute disseminated encephalomyelitis, and neuromyelitis optica. Multiple sclerosis, the most common of these diseases affecting over two million people worldwide and approximately 400,000 people in United States, is a chronic inflammatory disease, causing lesions and plaques of demyelination in the brain and spinal cord. However, none of the current clinical tests can confidently predict clinical multiple sclerosis progression or treatment efficacy due to a lack of sufficiently sensitive and effective biomarkers. T cells in multiple sclerosis patients display an activated phenotype with an increased avidity to myelin protein. As a result, we hypothesize that the avidity of autoreactive T cells is a disease marker that can be used to monitor disease progress, judge therapeutic response, or discover new biochemical disease markers for multiple sclerosis. However, none of the current technologies have achieved high-sensitivity and high-throughput measurement of cell avidity, in clinical samples, at the single cell level. The PI?s engineering advances in ?Acoustofluidics,? allowing for precise manipulation of single cells and liquids at unexpected resolution, shows promising potential for measuring the avidity of highly heterogeneous, clinical autoreactive T cells as well as for potentially sorting and analyzing single cell of interest, when combined with the PI?s ?High Throughput Microfluidic? technology. The objective is to create an acoustofluidic avidity cytometer to overcome the barriers in detecting clinical autoimmune disorders such as multiple sclerosis. However, there are several significant hurdles which include building a high-sensitivity and high-throughput avidity cytometer to profile single autoreactive myelin-specific T cells and determine their avidity distribution in clinical samples. Incorporating high-throughput microfluidics will allow the screening of single autoreactive T cells of interest for new autoantigens. The proposed instrumentation will potentially revolutionize the current clinical testing and discovery new markers for diagnosis, prognosis, and treatment efficacy of autoimmune disease including multiple sclerosis, rheumatoid arthritis, Crohn?s disease, etc.